Numerous hormones, drugs, and other agents do not cause their effects by penetrating into cells, but act through messenger systems across cell membranes. For many such effects the messengers are the cyclic nucleotides, cylic AMP and cylic GMP. By the use of derivatives of these compounds the membrane messenger generating system can be bypassed since derivatives can be selected which penetrate the membranes and mimic the effects of cyclic nucleotides in the cell. It is believed that the main intracellular target receptors for the cylic nucleotides are cyclic AMP- and cyclic GMP-dependent protein kinases which affect myriad enzyme systems through phosphorylation reactions. This laboratory and others have identified, purified, and characterized several of the protein kinases. Over the last few years in this laboratory, the mechanisms by which the cyclic nucleotides bind to and activate these enzymes have been investigated. Interestingly, the protein kinases possess two different kinds of cyclic nucleotide binding sites on each molecule. These two sites are stimulatory for each other and both are involved in enzyme activation. This proposal will utilize the knowledge gained in the previous grant period by testing the effects of cyclic nucleotide derivatives on intact fat cells, liver cells, H4 hepatoma cells and perfused hearts. Because of the presence of the two cyclic nucleotide binding sites, combinations of two derivatives, one selective for each site, will also be tested. The research should establish whether or not the cyclic AMP- or cyclic GMP-dependent protein kinase, and which isozyme, is responsible for a particular hormonal effect. Through competition, or other mechanisms, some of the derivatives could act as inhibitors rather than stimulators. A pathway of the action of insulin, another inhibitor of many cyclic nucleotide effects, will also be examined by the use of the derivatives. In summary, certain cellular processes in specific tissues can be examined and manipulated by the use of judiciously selected cyclic nucleotide derivatives which penetrate into cells quite well, are resistant to breakdown in the cell, and are potent in binding to one or the other of the two sites of one of the protein kinase isoenzymes. The cyclic nucleotide systems are basic to almost all normal and disease states. Of closest relevance are heart disease, diabetes, and defects in growth and differentiation.